1. LC-MS Based Metabolomics

2. Analysing the METABOLOME 1.Metabolite Extraction 2.Metabolite detection (with or without separation) 3.Data analysis

3. Metabolite Detection GC-MS: Naturally volatile or made volatile (any organic- flavors, sugars, lipids, acids) GC-MS: Naturally volatile or made volatile (any organic- flavors, sugars, lipids, acids) NMR – any compound containing hydrogen NMR – any compound containing hydrogen HP Liquid - Chromatography + detector HP Liquid - Chromatography + detector Comon detectors- Comon detectors- - UV-detector (phenolics) - MASS SPECTROMETER (MS) as detector (LC-MS)

4. Metabolite Detection MASS SPECTROMETER (MS) as detector (LC-MS): Compounds that are not well characterized by other methods: Non volatile High molecular weight Too sensitive to heat to be analyzed by GC

5. LC/MS Sample Introduction Sample Preparation Ions Detection Ions Separation Ionization MS Interface UV Spectra Separation (column) Efficient Gradient Your Sample Result LC- MS Today Data (computer)

6. Components in LC-MS Analyzer & Detector APcI ESI Compound ID Structure Elucidation Quantitation Ion Sorting Ion Formation Interface: Atmospheric Pressure Ionization Triple Quadrupole Quadrupole -Time Of Flight Quadrupole - Ion-Trap FT-MS Results LC Chemical Separation Software Peptide & Protein Sequencing

7. Mass Spectrometer 1.Breaks up constituents into molecular ions and other fragments 2. The ions then pass through an electric and/or magnetic field that separates them according to their mass-to-charge ratio (m/z) 3. Measures masses

8. Mass Spectrometer 4. Universal detection method * compared to UV/VIS (PDA), fluorescence etc. * more specific than NMR 5.More sensitive for most compounds 6. Structural information on metabolite * fragmentation pattern * accurate mass 7. For both LC and GC

9. Technology of LC-MS and LC-MS-MS – Interfaces- Ionization (elimination of solvent and generation of gas-phase ions) – e.g. Z Spray – Analyzers – Quadrupoles (Q) and Time of Flight (TOF)

10. LC-MS Interfaces Alternative Ionization Modes In MS- * Measuring the mass of a huge variety of compounds, in a huge variety of matrices * Need range of methods to IONISE all the different compounds

11. Alternative Ionization Modes EI or CI, Electron (impact) OR Chemical Ionization (in GC-MS) Gas-phase ionization methods Small volatile molecules are heated and enter the gas phase Not always suitable: Difficult to get large or involatile molecules into the gas phase Laser desorption Matrix-assisted laser desorption ionization (MALDI) Particle bombardment Fast atomic bombardment (FAB) Secondary ion mass spectrometry (SIMS) Field desorption Ionization

12. Alternative Ionization Modes EI or CI, Electron (impact) OR Chemical Ionization (in GC-MS) Gas-phase ionization methods Small volatile molecules are heated and enter the gas phase Not always suitable: Difficult to get large or involatile molecules into the gas phase Heating the non-volatile molecules degrades them

13. Alternative Ionization Modes Ionization for Non-Volatiles: Early ones- Particle bombardment Fast atomic bombardment (FAB) Secondary ion mass spectrometry (SIMS) Field desorption Ionization Thermospray ionization

14. Alternative Ionization Modes Ionization for Non-Volatiles: Early ones- Particle bombardment - Fast atomic bombardment (FAB) Secondary ion mass spectrometry (SIMS) single experiments, background signal from matrix Field desorption - complex, single experiments at once Thermospray - temprature degrades sample

15. Alternative Ionization Modes Atmospheric Pressure Ionization (API), in LC-MS Electrospray Ionisation (ESI): polar and semi-polar Atmospheric Pressure Chemical Ionization (APCI): less polar ES APCI lipids water polarity of analyte molecule

16. Atmospheric Pressure Ionisation (API) Techniques ESI and APCI differ in… How ions are generated ESI - solution phase ionization APCI - gas phase ionization Analyte compatibility ESI - polar compounds and large biomolecules APCI - less polar, smaller compounds (relative to those ionized by ESI) that have some volatility Flow rate compatibility ESI - 0.001 to 1 mL/min APCI - 0.2 to 2 mL/min ESI and APCI differ in… How ions are generated ESI - solution phase ionization APCI - gas phase ionization Analyte compatibility ESI - polar compounds and large biomolecules APCI - less polar, smaller compounds (relative to those ionized by ESI) that have some volatility Flow rate compatibility ESI - 0.001 to 1 mL/min APCI - 0.2 to 2 mL/min

17. Ionization Methods Electrospray (ESI) Atmospheric Pressure Chemical Ionization (APCI) Laser Desorption (MALDI) Fast Atom Bombardment (FAB or SIMS) Electron Impact Chemical Ionization “Soft” Ionization “Hard” Ionization ESI, APCI and MALDI can be used with LC EI ionization can be used with GC

18. How do the analytes become charged? - While in EI, loss of an electron producing a radical molecular ion - In soft ionisation techniques, analyte molecules are: protonated [M + H] or: de-protenoated [M - H] - Could also be sodiated, potassiated etc.. (adducts) - While in EI, loss of an electron producing a radical molecular ion - In soft ionisation techniques, analyte molecules are: protonated [M + H] + or: de-protenoated [M - H] - - Could also be sodiated, potassiated etc.. (adducts)

19. High positive or High negative charge Ion Formation in ESI

20. High postive or negative charge How do the analytes become charged? Reppeled positive (or negative) ions

21. Positive or Negative Modes? The formation of positive or negative ions depends on the sign of the applied electrical field ES+: (M+H) + Good ionization of basic compounds (get proton) E.g. amino, amide, ester, aldehyde/keto functional groups (formic acid in sample solution to help ionize) ES-: (M-H) - Acidic Compounds (give proton) E.g. organic acids, containing OH (ammonium buffer in sample solution to help ionize) The formation of positive or negative ions depends on the sign of the applied electrical field ES+: (M+H) + Good ionization of basic compounds (get proton) E.g. amino, amide, ester, aldehyde/keto functional groups (formic acid in sample solution to help ionize) ES-: (M-H) - Acidic Compounds (give proton) E.g. organic acids, containing OH (ammonium buffer in sample solution to help ionize)

22. Solvent Loss in ESI & Ion Formation High postive or negative charge

23. Electrospray Ionization ESI

24. Electrospray Theory

25. Summary ESI ESI is an atmospheric pressure ion source Small molecules singly chraged High MW samples become multiply chares (e.g. proteins) MWs of 150,000 Da (amu) cab be measured accurately ESI is an atmospheric pressure ion source Small molecules singly chraged High MW samples become multiply chares (e.g. proteins) MWs of 150,000 Da (amu) cab be measured accurately

26. Atmospheric Pressure Chemical Ionization - APCI Atmospheric Pressure Ionization Interface

27. APcI Source Ionization of solvent & solvent transfers the charge to analyte Sample is vaporised

28. APcI Theory

29. Atmospheric Pressure Chemical Ionisation (APcI) Atmospheric Pressure Chemical Ionisation (APcI) Low molecular weight (<1000 Da) Singly charged species Low molecular weight (<1000 Da) Singly charged species

30. ESI vs APcI TechniqueFlow RateMW RangeSpecies (ml/min)Produced ESI0.001 – 0.3<200,000 Da(M+H) + (M-H) - (M+nH) n+ (M+nH) n+ APcI0.2 – 2.0<1000 Da(M+H) + (M-H) -

31. Z SPRAY TM Source What happens from here?

32. Z-Spray Interface

33. Mass Analyzers Ion Sorting

34. Components in LC-MS Analyzer & Detector APcI ESI Compound ID Structure Elucidation Quantitation Ion Sorting Ion Formation Interface: Atmospheric Pressure Ionization Triple Quadrupole Quadrupole -Time Of Flight Quadrupole - Ion-Trap FT-MS Results LC Chemical Separation Software Peptide & Protein Sequencing

35. Quadrupole and Tandem Quadrupole Ion Separation Analyzers

36. Quadrupole Theory

37. Benefits of Time-Of-Flight MS high mass resolution (up to 10 or 5 ppm) high mass resolution (up to 10 or 5 ppm) exact mass exact mass

38. Resolution & Accuracy of a Mass-spectrometer

39. Resolution Resolution, (or Resolving Power ) of a mass sectrometer: A measure of its ability to separate adjacent ions At higher resolution, small differences may be detected.

40. Determining Resolution 2 adjacent ion peaks with a 10% valley max Double Ion method R = m ave mrmr Full Width at Half Maximum (50%, FWHM) or at 5% of the peak height Single Ion method R = m mm mrmr

41. Mass Analyzers Ion Cyclotron (FT-ICR-MS) Time of Flight (TOF) Magnetic Sector Quadrupole Ion Trap Quadrupole “High Resolution” Instruments “Low Resolution” Instruments

42. Low Resolution High Resolution High Resolution vs. Low Resolution 130 131 129

43. Resolution 249249.0700249.0580249.1479 3 different compounds Same nominal mass Low resolution 3 different compounds 3 different exact masses High resolution C 20 H 9 + C 19 H 7 N+ C 13 H 19 N 3 O 2 + C 20 H 9 +C 19 H 7 N+C 13 H 19 N 3 O 2 +

44. Mass Analyzers Ion Cyclotron (FT-ICR-MS) Time of Flight (TOF) Magnetic Sector Quadrupole Ion Trap Quadrupole Resolving Power Mass Accuracy 200,000 20,000 60,000 <1ppm 3-10ppm 2-5ppm 1,000 n/a

45. Mass Accuracy- FTMS - Instrument Calibration - 0.1353 148.060420 148.0604 Glutamate 0.0380 205.097192 205.0972 Tryptophan 0.0866 182.081184 182.0812 Tyrosine 0.1109 150.058317 150.0583 Methionine 0.1173 147.112783 147.1128 Lysine 0.0175 90.054998 90.0550 AlanineDifference(ppm)ExperimentalMassExactMassCalibrant

46. 1 5 12 2 1 0 1 0 0 150.0583257 5.826e-08 2 1 15 2 2 1 1 0 0 150.0586364 2.128e-06 3 3 15 0 2 0 1 0 1 150.0587525 2.902e-06 4 9 11 0 0 1 0 0 0 150.0592883 6.473e-06 5 3 15 0 1 2 0 1 0 150.0571936 7.486e-06 6 5 14 0 1 2 0 0 0 150.0595989 8.543e-06 7 4 16 1 0 1 0 0 1 150.0570349 8.544e-06 8 3 10 1 4 0 1 0 0 150.0569831 8.889e-06 9 2 16 3 0 2 0 0 0 150.0569188 9.318e-06 # 12C 1H 16O 14N 31P 32S 23Na 39K mass error 150.058317 Methionine C 5 H 12 NO 2 S 0.06 ppm Mass Accuracy Determining Empirical Formula, Structural Elucidation

47. Mass Accuracy Ability of a mass analyzer to assign the mass of an ion close to its true value (exact mass)  m accuracy = m real - m measured In ppm = 10 6 *  m accuracy / m measured  m accuracy

48. Mass Accuracy High mass accuracy (exact mass measurement) is usually associated to high resolution analyzers Unknown compound determination Exact mass helps to define its atomic composition

49. Scan Speed (or rate)  The rate at which we can acquire a mass spectrum, (mass units/sec).  Is an essential acquisition parameter for MS  Will affect the amount of information (qualitative and quantitative) that can reasonably be attained with a given mass analyzer.

50. Mass Analyzers

51. Next Class Data after ion detection in LC-MS